Glycosaminoglycans (GAGs) are involved in a host of regulatory processes centered in the extracellular matrix (ECM) of higher organisms. The extraordinary structural diversity of GAGs enables them to interact with a wide variety of biological molecules to modulate processes, including immune response and regulation of cell growth. GAG-pathogen interactions also affect most, if not all, of the key steps of microbial pathogenesis, including host cell attachment, invasion, cell-cell transmission, systemic dissemination, and evasion of host defense mechanisms. Additionally, much progress has been made in turning pathogens to therapeutics, specifically in the use of oncolytic viruses in cancer treatment. There is obvious interest in improving targeting to surface receptors up-regulated in cancer, including GAGs. Several characteristics of poxviruses make them well-suited for use as oncolytic virus therapeutics. In particular, poxviruses inherently have broad tumor tissue tropism. While they do not use GAGs for attachment or invasion, they do produce GAG-binding proteins for other purposes. The particular target of this study, VACV B18, is the type I interferon (IFN) binding protein (IFN/BP) secreted by the vaccinia virus (a member of the poxvirus family), that suppresses immune response through binding to IFN. This protein interacts with cell surface GAGs in order to extend the range of this suppressive effect over multiple cell surfaces. In particular, B18 has been shown to bind strongly to both heparin (HP) and heparan sulfate (HS); however, the structural basis for this recognition and binding remains unknown. This proposal will develop biochemical and solution NMR methods to address the structural factors and intermolecular interactions involved in B18-GAG binding. Understanding these specific interactions can lay the basis for manipulating GAG-protein interactions for therapeutic purposes. In order to determine the structural basis of GAG-B18 binding, a series of HS oligomers will be isolated using a combination of cell culture, isolation, digestion, and separation technology that allow preparation of isotopically labeled oligomers for structural study. With these in hand, existing solution NMR methods will be combined with novel sparse labeling and long range paramagnetic perturbation methods to determine structural models of the wild-type protein, both in it's free, unbound state, and in complex with specific HS oligomers. A comparison of the free and bound structures will illuminate the structural role of residues previously identified as important, and lead to the identification of HS fragment characteristics important for B18 binding. These advances will provide methods which will be applicable to other GAG binding proteins. Successful completion of this project will not only generate novel methodology, but also enhance molecular-level understanding of intermolecular B18-HS interactions in vaccinia virus and provide a molecular basis for the future design of HS analogs that can inhibit the immune suppressive action of B18.